The actions of hormones and drugs which inhibit adenylate cyclase in eukaryotic cells are mediated by specific receptors and an inhibitory GTP-binding regulatory protein (Gi). Bordetella pertussis toxin (PT) catalyzes the ADP-ribosylation of a 41,000 dalton peptide believed to be a subunit of Gi and abolishes the effects of inhibitory agonists. In studying the effects of PT on opiate receptors in neuroblastoma x glioma (NG108-15) hybrid cells, we found that binding of 3-H-enkephalinamide, an opiate agonist, was reduced 90% in membranes incubated with PT. Furthermore, the receptors' responsiveness to guanyl nucleotide, a hallmark of receptor-Gi interaction, was reduced. The number of opiate receptors assessed by binding of 3-H-naloxone, an opiate antagonist, was unaltered by PT. These data are consistent with the hypothesis that PT-catalyzed ADP-ribosylation impairs the interaction of Gi with the inhibitory receptor-ligand complex, effectively uncoupling the inhibitory receptor from Gi and the cyclase catalytic unit.